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Question 1

Introduction

Answer 1

• Transcriptional activation is central to cell fate regulation.
• The process is controlled by chromatin accessibility at regulatory loci such as promotors and enhancers.
• The epigenetic mechanisms that contribute to cellular differentiation and identify formation during organ development and in disease are under intense investigation.

Question 2

Single Cell Approaches

Answer 2

• Single cell RNA sequencing technologies enable the measurement of transcriptome profiles at unprecedented scale and resolution.
• SC-ATAC-seq works by using a hyperactive Tn5 transposase enzyme to insert sequencing adapters into accessible regions of chromatin. After sequencing, regions with inserted adapters represent open chromatin, providing insights into gene regulation
• Combining these technologies provides insight into the epigenetic landscape of heterogeneous cell population.
  Interpreting these data is a huge challenge.
• The enormous data sets these technologies provide have unmatched power to inform – but also to confuse.
• Large scale computation methods have been designed to do so – though over 1,400 tools are now available to analyse scRNA-seq data alone. But still challenging.
• Also, an inadvertent potential for bias via normalisation of data and annotation of clusters can skew results.
  o Not to mention the challenges of linking cluster to function.
• This highlights one exciting future application for AI.

Question 3

Kidney Development

Answer 3

• Transcriptional activation is central to the processes regulating renal cell fate and heavily influenced by polycomb repressive complex function.
• Polycomb group proteins are present on repressed genes.
• The complexes regulate gene expression by repressing key developmental genes, influencing cell fate by maintaining cell identity and controlling differentiation processes.
• They modify histones by adding methyl or ubiquitin groups, leading to chromatin compaction and hence inhibition of gene transcription.

Question 4

Modelling the Kidney

Answer 4

• Modelling the kidney is very challenging owing to the organs complex organisation.
• Organoids are miniature models derived from stem cells that exhibit remarkable similarities with native tissues in terms of cell type, morphology and function – making them ideal for regenerative medicine investigations.
• The models can be adult stem cell derived, or iPSC derived.

Question 5

Protocols

Answer 5

• Two ground-breaking kidney organoid protocols were published in 2015.
• The takasato protocol resembled the first trimester of fetal kidney tissue and had 21% non-kidney cells present.
• The Morizane lab utilised a lower dose of fibroblast growth factor, and their organoids contained more complex compartmentalisation. These had 11% non-renal cells. .
• Hence, neither were perfect.
• Positive feedback of SC-data has informed the elimination of most off-target muscle and neuronal cells.
  o For example, provided insights that inhibiting BDNF-NTRK2 signalling reduces off target cell types by 90%

Question 6

Establishing and validating a model

Answer 6

• Validation pertains to identity, of which SC omics can inform us.
• Ultimately, the best comparison between a human kidney, and a kidney model that were designed in a dish – is a human kidney.
• Hence, comparisons between single cell human data and single cell organoid data informs everything that is done in this field.
• But this is difficult.
• Sc-tech enable tracking the epigenetic remodelling of individual cells over time.
• Researchers can elucidate the regulatory networks governing cell fate decisions and tissue patterning within organoid cultures.

Question 7

Disease Modelling

Answer 7

• Organoids provide a fantastic platform for disease modelling, and sc-approaches provide huge insight into disease processes.
• iPSCs also allow for patient specific disease modelling.
• Crean et al., recently modelled kidney fibrosis in a takasata organoid, with profibrotic factor TGFB1.
• This unveiled two novel fibroblast populations, alongside novel regulators of fibrosis, linked to dynamic epigenomic changes.
• The novel populations highly resembled activated-myofibroblasts seen in vivo, in fibrosis.
• There were some off target populations, in keeping with the protocol.
• The group has since improved the extracellular environment the organoids are grown in allowing for better control of tissue morphogenesis.
• And are looking at introducing vascularisation using chips.
• All informed by their sc analysis.
• But once we have our model, we can induce the changes, like with creans TGFB – watch the cells adopt their new activate myofibroblast states.

o Pseudotime analysis will reveal the lineage relationships of each cell type and allow us to identify cell fate decisions.
o Mapping these inflection points by transcriptional profiling and accessibility will establish a hierarchy of cell fate.
o Importantly, it will provide us with the druggable opportunity we are seeking.

Question 8

Regenerative medicine

Answer 8

• Chronic kidney disease is a global public health problem, with an increasing incidence.
• There is only symptomatic treatment available, fervent global unmet need for disease modifying.
• The goal of regenerative medicine is to replace or regenerate damaged human cells, tissues or organs in order to completely restore or establish their normal structures and functions.
• Therefore, the generation of these organoids holds great promise as an innovative source of function nephrons for CKD patients – though challenges remain.
• Off target populations need to be reduced, which is happening as discussed.
• Implementation of self-organisation in the organoids is challenging but again - is being addressed.
  o Incorporating steering cues such as hydrogels drive the organisation.
• Maturation is probably the biggest challenge. For example, no kidney to date shows an established ureteric epithelium or a patent vasculature.
  o SC-omics providing molecular insights into developmental mechanisms which can in turn to used to optimise protocols.
• Vascularisation will also likely play a central role.

Question 9

Outro

Answer 9

• From my research, I believe we are very long way off a functional kidney, that one could transplant. Or even epigenetic based therapeutics, in which we could reverse damage, like Sinclair and his mice.
• But just because we’ve a long way to go, doesn’t mean achieving this will take a huge amount of time.
• The human genome project was completed in 2003. It took 13 years to map. With our current technology – we can do it in a few days.
• We have come so far with our molecular technologies, and single cell approaches are a game changer.
• We just need to find out the cause of fibrosis or age or whatever we want to solve, find out how we get from fate A to fate B – in detail. What enhancers are open, closed, expressing. What super enchancers are on and so forth. And then, we have our target. And then our distant future is a therapeutic.